Refinement ends up with a feature-less ball

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Hi everyone,
I converted the post-refinement results from relion and was trying to switch to frealign.
First I use the conversion script to convert relion star file to par file.(http://grigoriefflab.janelia.org/frealign_conversion_scripts)
Then I use relion_stack_create to generate the particle stack.

Frealign could run and finish without error (V9.11).
However, no matter how I changed the parameter, I could only get a feature-less ball.

I am not sure which step went wrong.
Any input will be greatly appreciated.

Below is my mparameters file.

Control parameter file to run Frealign
======================================

This file must me kept in the project working directory from which the refinement scripts are launched.

Note: Please make sure that project and scratch directories (if specified) are accessible by all sub-processes that are run on cluster nodes.

# Computer-specific setting
cluster_type none ! Set to "sge", "lsf", "slurm", "stampede", "pbs" or "condor" when running on a cluster, otherwise set to "none".
nprocessor_ref 64 ! Number of CPUs to use during refinement.
nprocessor_rec 8 ! Number of CPUs to use during reconstruction.
mem_per_cpu 8192 ! Memory available per CPU (in MB).

# Refinement-specific parameters
MODE 1 ! 1, 2, 3 or 4. Refinement mode, normally 1. Set to 2 for additional search.
start_process 2 ! First cycle to execute. Output files from previous cycle (n-1) required.
end_process 10 ! Last cycle to execute.
res_high_refinement 10.0 ! High-resolution limit for particle alignment.
res_high_class 8.0 ! High-resolution limit to calculate class membership (OCC).
nclasses 1 ! Number of classes to use.
DANG 200.0 ! Mode 3, 4 and 5: Angular step for orientational search.
ITMAX 200 ! Mode 2 and 4: Number of repetitions of grid search with random starting angles.

# Dataset-specific parameters
data_input Data ! Root name for parameter and map files.
raw_images stack.mrc
image_contrast P ! N or P. Set to N if particles are dark on bright background, otherwise set to P.
outer_radius 180.0 ! Outer radius of spherical particle mask in Angstrom.
inner_radius 0.0 ! Inner radius of spherical particle mask in Angstrom.
mol_mass 400.0 ! Molecular mass in kDa of particle or helical segment.
Symmetry C4 ! Symmetry of particle.
pix_size 2.6 ! Pixel size of particle in Angstrom.
dstep 10.0 ! Pixel size of detector in micrometer.
Aberration 2.7 ! Sherical aberration coefficient in millimeter.
Voltage 300.0 ! Beam accelleration voltage in kilovolt.
Amp_contrast 0.07 ! Amplitude contrast.

# Expert parameters (for expert users)
XSTD 0.0 ! Tighter masking of 3D map (XSTD > 0) or particles (XSTD < 0).
PBC 2.0 ! Discriminate particles with different scores during reconstruction. Small values (5 - 10) discriminate more than large values (50 - 100).
parameter_mask "1 1 1 1 1" ! Five flags of 0 or 1 (e.g. 1 1 1 1 1). Determines which parameters are refined (PSI, THETA, PHI, SHX, SHY).
refineangleinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles.
refineshiftinc 4 ! When larger than 1: Alternate between refinement of OCC and OCC + angles + shifts.
res_reconstruction 5.2 ! High-resolution limit of reconstruction. Normally set to Nyquist limit.
res_low_refinement 200.0 ! Low-resolution limit for particle alignment. Set to particle dimention or larger.
thresh_reconst 0.0 ! Particles with scores below this value will not be included in the reconstruction.
thresh_refine 50.0 ! Mode 4: Score threshold above which search will not be performed.
nbootstrap 1000 ! Number of bootstrap volumes to calculate real-space variance map.
FMAG F ! T or F. Set to T to refine particle magnification. Not recommended in most cases.
FDEF F ! T or F. Set to T to refine defocus per micrograph. Not recommended in most cases.
FASTIG F ! T or F. Set to T to refine astigmatism. Not recommended in most cases.
FPART F ! T or F. Set to T to refine defocus for each particle. Not recommended in most cases.
FFILT T ! T or F. Set to T to apply optimal filter to reconstruction. Recommended in most cases.
FMATCH F ! T or F. Set to T to output matching projections. Only needed for diagnostics.
FBEAUT F ! T or F. Set to T to apply symmetry also in real space. Not needed in most cases.
FBOOST T ! T or F. Set to T to allow potential overfitting during refinement. Not recommended in most cases.
RBfactor 0.0 ! B-factor sharpening (when < 0) applied during refinement. Not recommended in most cases.
beam_tilt_x 0.0 ! Beam tilt in mrad along X-axis.
beam_tilt_y 0.0 ! Beam tilt in mrad along y-axis.
mp_cpus 1 ! Number of CPUs to use for each reconstruction job.
restart_after_crash F ! T or F. Set to T to restart job if it crashes.
delete_scratch T ! Delete intermediate files in scratch directory.
qsub_string_ref "" ! String to add to cluster jobs submitted for refinement (only for SGE, SLURM and PBS clusters).
qsub_string_rec "" ! String to add to cluster jobs submitted for reconstruction (only for SGE, SLURM and PBS clusters).
first_particle
last_particle
frealign_bin_dir
scratch_dir

# Masking parameters (for expert users)
mask_file
mask_edge 5 ! Width of cosine edge in pixels to add around mask. Set to 0 to leave mask unchanged.
mask_outside_weight 0.0 ! Factor to downweight density outside of mask (normally 0.0 - 1.0).
mask_filt_res 0.0 ! Filter radius (in A) to low-pass filter density outside density. Set to 0.0 to skip filtering.
mask_filt_edge 5 ! Width of cosine edge in reciprocal pixels to add to filter function.

hi qwertgfdsa,

If you do a simple frealign_calc_reconstructions, do you get a featureless ball?
This would indicate that something went wrong during conversion, probably during generation of the mrc stack.
How do you do this step?

Best,

Axel

In reply to by Axel

Hi Axel,
realign_calc_reconstructions of cycle 1 give me a normal map.

I figured it out why.
Just a stupid mistake.
My particle has a diameter of 180A
but I input 180 as the outer_radius....

Thank you very much for the help!